Project Summary Centromeres are essential chromosomal elements that mediate kinetochore assembly and accurate chromosome segregation. Centromere defects lead to chromosome missegregation, with detrimental effects on cell and organism health and fertility. In most multicellular species, centromeres are composed of large regions of highly repetitive DNA marked by chromatin containing the centromere-specific histone variant CENP-A. Previous work demonstrated that both centromeric DNA and CENP-A chromatin have the potential to initiate centromere activity de novo; however, their respective contributions to centromere specification in mitosis and meiosis have remained elusive. The overall goal of this proposal is to determine how centromeric DNA and chromatin contribute to centromere identity. The centromeres of metazoans have been refractory to full sequencing and assembly due their large size and highly repetitive nature, hampering our ability to systematically interrogate the role of centromeric DNA elements. Additionally, there are currently no systems in which to test if de novo centromeres, which are devoid of centromeric DNA, and can sustain centromere function and specification through mitotic and meiotic divisions. Using our unique advancements in Drosophila, which include the identification and assembly of its centromeric sequences and the establishment of an inducible de novo centromere system, this proposal will: 1) test if chromatin-mediated centromeres can sustain chromosome segregation through development and meiosis, effectively replacing endogenous centromeres; 2) test specific hypothesis on how centromeric DNA elements may contribute to CENP-A chromatin establishment or maintenance. Collectively, this work will shed light into centromere specification mechanisms in Drosophila, an exceptional model system that allows centromere studies in the context of animal development and fertility- with broad relevance to other species, including humans.